The biomarkers’ landscape of post-COVID-19 patients can suggest selective clinical interventions

In COVID-19 clinical symptoms can persist even after negativization also in individuals who have had mild or moderate disease. We here investigated the biomarkers that define the post-COVID-19 clinical state analyzing the exhaled breath condensate (EBC) of 38 post COVID-19 patients and 38 sex and age-matched healthy controls via nuclear magnetic resonance (NMR)-based metabolomics. Predicted gene-modulated microRNAs (miRNAs) related to COVID-19 were quantified from EBC of 10 patients and 10 controls. Finally, clinical parameters from all post-COVID-19 patients were correlated with metabolomic data. Post-COVID-19 patients and controls showed different metabolic phenotype (“metabotype”). From the metabolites, by using enrichment analysis we identified miRNAs that resulted up-regulated (hsa-miR146a-5p) and down-regulated (hsa-miR-126-3p and hsa-miR-223-3p) in post-COVID-19. Taken together, our multiomics data indicate that post-COVID-19 patients before rehabilitation are characterized by persistent inflammation, dysregulation of liver, endovascular thrombotic and pulmonary processes, and physical impairment, which should be the primary clinical targets to contrast the post-acute sequelae of COVID-19.


NMR-based metabolomics of patients' EBC
To define the post-COVID physiological state, we profiled by NMR the EBC from patients, and compared them with the corresponding profiles of healthy subjects.Figure S1 compares the NMR spectra of the EBC samples from a healthy subject (a) with that of a patient (b), and resonances' assignments are reported in Table S1.Notably, saliva contamination was absent in both samples as the most intense saliva signals, originating from carbohydrates and resonating between 3.3 and 6.0 ppm, are absent.PCA was used to explore data trend and possible outliers (data not shown).We then carried out supervised OPLS-DA, which yielded a regression model with high-quality parameters (R 2 = 0.81, Q 2 = 0.87 and CV ANOVA p = 2.3 × 10 −12 ), and a clear class discrimination (Fig. 2).In the associated loadings plot (not shown), the post-COVID group, with respect to controls, presented upregulation of ethanol, lactate and acetoin, and downregulation of acetate, acetone, fatty acids, isocaproate, isovalerate, methanol and valerate.Their statistical significance is reported as box and whiskers plots in Supplementary Figs.1-3.These results indicate that patients present a metabotype completely different from that of healthy subjects.
The discriminating biomarkers were used to identify the metabolic networks altered in post-COVID.Application of enrichment metabolic analysis indicated the potential biological mechanisms producing the separation between post-COVID and controls.With a threshold of p < 0.05, we uncovered synthesis and degradation of ketone bodies, pyruvate metabolism, propanoate metabolism, butanoate metabolism, cAMP signaling pathway, inflammatory mediator regulation of TRP channels and carbon metabolism as the most probable activated pathways.They mark the differences between the post-COVID-19 metabotype with respect to controls.The results of the enrichment analysis are reported as Supplementary Table S2.

miRNA analysis
Potential genes related to altered metabolites found in EBC were derived from gene-metabolite interaction network analysis (Supplementary Table S3).Putative miRNAs involved in the modulation of the found genes were uncovered by an in silico analysis using the miRNet tool.This approach integrated the metabolomic analysis and miRNAs modulation in the same samples.Enrichment analysis based on the hypergeometric test explored 20 miRNA functions significantly modulated (p < 0.05).Validation of miRNAs through qRT-PCR was obtained considering the functions cell cycle (74 hits, Gene ontology (GO) annotations number GO:0007049), regulation of stem cell proliferation (74 hits, GO:0072091), cell death (73 hits, GO:0008219), aging (70 hits, GO:0007568), hematopoiesis (68 hits, GO:0030097) and angiogenesis (66 hits, GO:0001525) (Supplementary Table S4).

Correlation of EBC metabolites with clinical parameters
The post-COVID-19 metabolites from EBC were associated with the clinical parameters obtained at the hospitalization before rehabilitation.The heatmap in Fig. 4 shows the significant Pearson correlation coefficients (p < 0.05) between the metabolites and at least one clinical parameter.Considering a threshold value of ρ ≥ |0.5|, we identified a positive correlation of 0.7 between propionate/isobutyrate (label 2 in Fig. 4) and creatinine (dark blue box with a red double asterisk, see the color code in Fig. 4).Positive correlations of 0.5 were observed between acetoin (label 1) and propionate/valine (label 3) with creatinine, isobutyrate (label 5) and alanine aminotransferase (ALT), lactate (label 10) and pH, glycine (label 21) and leukocytes, 3-hydroxyisobutyrate (label 22) and leukocytes, and ethanol (label 23) with platelets (blue boxes with a red asterisk).Negative correlations of − 0.7 were observed between acetoin, propionate/isobutyrate and propionate/ valine with FEV 1 /FVC (labels 1, 2 and 3, respectively, pale yellow boxes with a black double asterisk).Negative  correlations of − 0.5 involved methanol (label 6) with weight and the six-minute walking distance (6MWD), acetone (label 18) and 6MWD, and glycine (label 21) and pH (light green boxes with a black asterisk in Fig. 4).Positive correlation indicates similar behavior between metabolites and clinical values (increase/increase, decrease/ decrease), while negative correlation refers to opposite behavior (increase/decrease, decrease/increase).Such correlations indicate that clinical parameters can be monitored via metabolites, which could become noninvasive markers of the clinical status.

Rehabilitation of post-COVID-19 patients: analysis of the clinical data between admission and discharge
The effects of rehabilitation on patients were evaluated by comparing the clinical/laboratory data of each patient at the admission in an average rehabilitation cycle of 24.3 days (in) (Table 1) and at discharge (out).The scores multilevel PLS-DA plot of Fig. 5 shows that the discharge status (black dots, out) is different from the one at the admission (red dots, in).In particular, at the admission, patients presented higher values of creatine, triglycerides (TGs), leukocytes, urea, red blood cell count, systolic blood pressure, total cholesterol (TC), platelets, hematocrit, weight, diastolic blood pressure, hemoglobin, glycemia, C-reactive protein (CRP), ALT, D-dimer, aspartate aminotransferase (AST), FEV 1 /FVC and CAT.At discharge, patients were characterized by higher values of pH, total lung capacity (TLC), HCO 3 , uricemia, albumin, PaCO 2 , DLCO/VA, DLCO, SpO 2 , Barthel, PaO 2 , FEV 1 %, FVC, FVC%, FEV 1 and 6MWD.This is depicted in Fig. 6, which reports the contribution plot related to the  above multilevel PLS-DA model, where each bar represents the loadings value for each variable on the principal component PC1 at the admission (red bars) and at discharge (black bars).Statistical significance was found for AST, ALT, D-dimer, CAT, Barthel, DLCO, SpO 2 , PaO 2 , FEV 1 /FVC, FEV 1 , FVC, FEV 1 %, FVC% and 6MWD (Table 1), which are the principal clinical parameters that are carried over upon negativization.Therefore, post-COVID-19 patients should be monitored for liver damage (AST and ALT), endovascular thrombotic processes (D-dimer), persisting pulmonary symptoms (CAT, Barthel, DLCO, SpO 2 , PaO 2 , FEV 1 , FVC, FEV 1 /FVC, FEV 1 %, FVC%), and physical impairment (6MWD).The relationship between the above parameters and the statistically significant EBC metabolites (top arrows in Fig. 4) indicated negative correlations between increased acetoin (label 1) and decreased FEV 1 /FEV (ρ = − 0.7, underlined in Fig. 4), decreased methanol (label 6) and acetone (label 18) with increased 6MWD, increased ethanol (label 23) and D-dimer (all presenting ρ = − 0.5).www.nature.com/scientificreports/Taken together, the metabolomic, the miRNAs and the clinical data point out that post-COVID patients still present dysregulation of the liver, endovascular and pulmonary parameters.

Discussion
Our results show that post-COVID-19 patients present several dysfunctions from which post-acute sequelae could originate.In particular, the post-COVID-19 group showed persistent lung inflammation as indicated by upregulation of ethanol, lactate and acetoin, and downregulation of acetate, methanol, acetone, fatty acids, isocaproate, isovalerate and valerate.In fact, increased acetoin level is associated with airway inflammation 19 , and reduction of methanol was observed in the EBC of lung cancer patients 20 .Short-chain fatty acids acetate, isovalerate, valerate and isocaproate (SCFAs) are involved in the regulation of several leukocyte functions linked to the production of cytokines, eicosanoids and chemokines, and are reported to affect leukocyte migration to the inflammation foci 21 .Acetone and lactate were detected in the bronchoalveolar lavage fluid of cystic fibrosis patients with varying levels of inflammation 22 .In addition, lactate excess can bring about a noticeable raise in ROS and apoptosis in A549 alveolar cells 23 .It was reported that non-survivor COVID-19 patients had higher lactate levels with respect to survivors at the intensive-care unit admission 24 .Furthermore, lactate is the main downgrading product of anaerobic metabolism, and it is well known that COVID-19 patients present hypoxic lung damage and respiratory failure, and that hypoxia is an indicator of COVID-19 mortality 25 .Significantly different concentrations between COVID-19 patients within 21 days from clinical diagnosis and post-COVID-19 groups were observed for acetate, acetone and lactate also in plasma 26 .
Correlation of EBC metabolites with clinical data from patients showed statistically significant relationships between increased acetoin and reduced FEV 1 /FVC (ρ = − 0.7), decreased methanol and acetone with increased 6MWD (ρ = − 0.5), and increased ethanol and decreased D-dimer (ρ = − 0.5), which indicate that these metabolite alterations are manifestations of the corresponding physiological functions.As a confirmation, reduction of methanol and acetone and the corresponding 6MWD increase was observed in chronic obstructive pulmonary disease (COPD) patients after a 5-week rehabilitation program 27 , and ethanol can reduce the global fibrinolytic capacity of whole blood, measured as D-dimer production during incubation of blood clots 28 .
From the above metabolites we identified the most probable dysregulated metabolic pathways, namely synthesis and degradation of ketone bodies, pyruvate metabolism, propanoate metabolism, butanoate metabolism, cAMP signaling pathway, and inflammatory mediator regulation of TRP channels.Interestingly, upregulation of ketone bodies and pyruvate metabolisms has been observed in previous NMR-based metabolomics studies of serum/plasma samples from post-COVID patients 26,[29][30][31] .
Ketone bodies (KBs) are produced by hepatocytes' mitochondria where fatty acids enter upon adipocytokine signaling.Interestingly, two adipocytokines, IL-6 and tumor necrosis factor-alpha (TNFα), are related to COVID-19 severity and patients' death 32 .Degradation of KBs (ketolysis) implies elevated levels of KBs in the blood and urine (ketosis).Ketosis shows an anti-inflammatory activity since β-hydroxybutyrate (β-HB), derived from the KB acetoacetate, is a key regulator of inflammation pathways like the NLRP3 inflammasome 33 .It has been suggested that in SARS-CoV-2 infection, treatments increasing β-HB levels could improve host defenses against respiratory viral infection while decreasing inflammation 34 .Additionally, the high levels of triglycerides and triglycerides-rich lipoproteins observed in COVID plasma 26 could be generated by a limited oxidation of acetyl-CoA inside the mitochondria, therefore favoring the synthesis of ketone bodies and the high levels of β-HB, acetoacetate and acetone in COVID-19 patients 35 .
cAMP is involved in several inflammatory pathways, being able to inhibit ROS generation and proinflammatory cytokine production, primarily IL-6 and TNF-α 36 .Furthermore, preserving the cAMP concentration in the pulmonary tissue can improve lung functions 36 , which are essential in COVID-19 patients.Interestingly, anosmia and ageusia, which have been observed in COVID-19 patients, have also been related to the intracellular levels of cAMP 37 .
The propanoate and butanoate metabolisms describe the metabolism of the SCFAs propionate and butyrate.SCFAs mediate the communication between the intestinal microbiome and the immune cells via free fatty acid receptors (FFARs), and dysregulation of the FFAR2/3 receptors' expression favored the insurgence of respiratory diseases 38 .We have observed that post-COVID 19 patients showed, with respect to controls, alteration of acetate, fatty acids, isocaproate, isovalerate, valerate (all SCFAs), and fatty acids, which are involved in the production of cytokines, eicosanoids, and chemokines responsible for the lung hyperinflammation in severe COVID-19 patients 39 .
Transient receptor potential (TRP) channels are widely expressed in tissues that are infected by SARS-CoV-2 and have been proposed as targets for adjuvant therapies against COVID-19 40 .Most of the clinical manifestations of COVID-19 activate different TRP channels.For example, TRPV4 is involved in the recruitment of neutrophils and macrophages during lung injury 41 and relates to hearing loss/impairment 40 .Loss of either TRPM4 or TRPM5 channels may significantly impair taste 42 and olfaction 43 .TRP channels also contribute to several cardiac complications (arrhythmias, cardiac fibrosis and myocyte hypertrophy) observed in COVID-19 patients 44 .
Using miRNet, from the discriminating metabolites we identified the perturbed genes, which in turn prompted the miRNAs altered in EBC.miRNAs have emerged as regulators of COVID-19 45,46 .In particular, we found hsa-miR-126-3p and hsa-miR-223-3p downregulated in post-COVID-19, while hsa-miR-146a-5p was upregulated.They are involved in the regulation of ACE2, the binding site of the virus, and in the inflammatory responses and immune regulation 47 .hsa-miR-126-3p attenuates lung inflammation via different pathways that reduce many proinflammatory cytokines including IL-6 48 , which in COVID-19 has been linked to high mortality risk 32 .In COVID-19 patients, the serum level of hsa-miR-126-3p was considerably reduced with the increase of disease grade 49 , and this pattern was also observed in patients non-responsive to therapies 50 .hsa-miR-126-3p downregulation was also detected in plasma samples of COVID-19 patients with respect to a healthy control www.nature.com/scientificreports/group, while no downregulation was observed between severe and mild patients 51 , which was instead previously reported 52 .Furthermore, a positive correlation between miR-126-3p and neutrophils levels, and a significant negative correlation with IL-6 and D-dimer were observed 53 .Interestingly, in vitro hsa-miR-126-3p exhibited neutralizing activity against SARS-COV-2 infection 49 .We here found that hsa-miR-126-3p does not return to the pre-COVID-19 values, and this is an indication of the persistent inflammation status after negativization.hsa-miR-126-3p also shows a pro-angiogenic role by stimulating endothelial cell proliferation 54 .The post-acute COVID-19 syndrome is associated with a persistent endothelial dysfunction, directly correlated with the severity of pulmonary impairment 55 , whose recovery is normally related to maintaining the physiological endothelial functions.Therefore, in line with the above results, the decrease we observed for hsa-miR-126-3p suggests the persistent presence of endothelial damage in patients.
Serum hsa-miR-223-3p directly inhibits the viral S protein expression and SARS-CoV-2 replication 56 , and is implicated in the regulation of inflammatory responses by inhibiting the action of the NLRP3 inflammasome and modulating the expression of inflammatory chemokines and cytokines 57 .In a possible mechanism, decrease of the has-miR-223-3p expression should increase NLRP3 expression levels and promote pyroptosis 58,59 .Furthermore, serum miR-223-3p Therefore, the reduced level of hsa-miR-223-3p observed in post-COVID-19 patients confirms that inflammation is still present after negativization.Interestingly, hsa-miR-223-3p was amplified by long-term physical exercise 56 , and we here found that 6MWD is the most important factor that characterizes the hospital discharge after post-COVID-19 rehabilitation.Taken together, this suggests a beneficial action of hsa-miR-223-3p with the consequent reduction of inflammation 56 .
Upon a viral infection, has-miR-146a is primarily produced to regulate the innate immune response and inflammation by negatively regulating the NF-κB pathway 60,61 .Therefore, its expression in COVID-19 decreases inflammatory disorders in target organs such as the lungs, heart, brain, skin, and underlying vascular disease 61,62 .The hsa-miR-146a-5p increase we observed in post-COVID-19 patients is an indication of the path to recovery, as the levels of IL-1, IL-6 and TNF-α cytokines are inversely correlated to has-miR-146a production 63,64 .In fact, hsa-miR-146a-5p was found ca.threefold higher in a COVID-19 post-acute group than in the acute group 65,66 , and COVID-19 patients who did not respond to tocilizumab treatment presented a reduction of has-miR-146a-5p with respect to responders, and its reduction in non-responders was associated to a higher risk of adverse outcomes 53 .
The above miRNAs are involved in cell cycle, regulation of stem cell proliferation, cell death, aging, hematopoiesis, and angiogenesis functions.Although nonspecific, cell cycle, regulation of stem cell proliferation and cell death could reflect the impact of COVID-19 on several multiorgan cellular processes, in line with the results of a proteomic analysis of autoptic samples from seven organs in COVID-19 patients 67 .Furthermore, the regulation of stem cell proliferation promotes remodeling and lung tissue regeneration after COVID-19-induced pneumonia and can help patients' recovery 68 .Similarly, for cell death, acutely ill COVID-19 patients revealed an upregulation of cell death programs genes, acting in tissue specific manner 69 .
More specific are aging, hematopoiesis and angiogenesis.Aging is a main risk factor for severe COVID-19 and its worst outcomes because it induces immunosenescence, which hampers the response to the virus 70 , and inflammaging, a low-grade diffused inflammation 71 .Hematopoiesis alteration is associated with severe and fatal COVID-19 as SARS-CoV-2 alters the bone marrow microenvironment, weakening hematopoiesis and causing hemocytopenia 72 .Furthermore, IL-6, which increases dramatically in COVID-19, is important for regulation of hematopoiesis as it stimulates the production of bone marrow neutrophils 73 .Regarding angiogenesis, autoptic lungs from patients died from SARS-CoV-2 infection indicated the presence of significant new vessel growth and a corresponding differential upregulation of angiogenesis-associated genes 74 .Such a compensatory angiogenesis mechanism was also observed in heart, liver, kidney, brain and lymphoreticular organs in patients who died from COVID-19 75 .
Comparing clinical data from post-COVID-19 patients before and after the admission in a rehabilitation cycle, we detected dysregulation of parameters related to liver damage (AST and ALT), endovascular thrombotic processes (D-dimer), persisting pulmonary symptoms (CAT, Barthel, DLCO, SpO 2 , PaO 2 , FEV 1 , FVC, FEV 1 /FVC, FEV 1 %, FVC%), and physical impairment (6MWD).SARS-CoV-2-infected subjects present alterations of liver biochemistry 76 .Since AST and ALT increase is associated with the reduction of peripheral oxygen saturation in viral pneumonias 77 , it is expected that systemic hypoxia in COVID-19 may also alter AST and ALT levels.In fact, a five-fold increase of AST and ALT levels in COVID-19 with respect to normal is associated with an increased risk of death 78 , causing elevated levels of CRP (which is synthesized by the liver), D-dimer, ferritin and IL-6 76 .Therefore, the increased CRP values in patients before rehabilitation again confirms the persistence of liver alteration and inflammation.
Both venous and arterial thromboses characterize COVID-19 pathology 79 .D-dimer is an indirect marker of active coagulation and thrombin formation, and represents a mirror of the endovascular thrombotic processes.Higher levels of D-dimer are observed in severe patients infected with SARS-CoV-2 compared to nonsevere ones, and, significantly, increased D-dimer has been reported in COVID-19 nonsurvivors with respect to survivors, and the concentration continues to rise until death 80 .
The following limitations of the study should be considered.First, although the number of enrolled patients encompasses that indicated by backward analysis, our results depend on a relatively limited number of subjects (38 patients and 38 controls).For this reason, we combined different types of biomarkers (EBC, miRNAs and clinical parameters), which represent complementary physiological aspects.Second, the patients were not consecutively recruited since they were selected from those of the rehabilitation division.As such, parameters like hospitalization for acute cases and rehabilitation period were variable, ranging between 7 and 50 days, and 5 and 57 days, respectively.Furthermore, only 3 females (8%) were comprised in each group because the patients admitted were typically males.Also, the possibility that conditions/treatments not recorded because of the flexibility at the hospital admission affects our conclusions cannot be excluded.We are aware that such uncontrolled

Study procedures
After signing the informed consent, all convalescent COVID-19 patients underwent a detailed collection of key demographic and clinical information related to the acute phase of COVID-19, lung function, physical performance, comorbidities and treatment(s).Following the same exclusion criteria as convalescent COVID-19 patients, data were extracted from an irreversibly de-identified electronic dataset for control subjects.Venous blood samples were used for the common hemato-chemical parameters.Arterial blood samples were collected to measure oxygen (PaO 2 ) and carbon dioxide tension (PaCO 2 ) using a blood gas analyzer (ABL 825® FLEX BGA, Radiometer Medical Aps, Copenhagen, Denmark).According to the protocols of the Spirometry parameters and diffusion lung capacity for carbon monoxide (DLCO) were also evaluated with an automated equipment (Vmax® Encore, Vyasis Healthcare, Milan, Italy) as reported 82,83 .Forced expiratory volume in 1 s (FEV 1 ), forced vital capacity (FVC) and DLCO were expressed both as numerical values and percentages of predicted values (FEV 1 %, FVC% and DLCO%, respectively).The COPD Assessment Test (CAT) 84 and the Barthel index were also administered to patients to evaluate the impact of the disease on daily living.Exercise capacity was tested by measuring the 6MWD 85 .All the clinical and the instrumental analyses were carried out at the admission (in) and at the discharge (out) after rehabilitation.

Rehabilitation
The rehabilitation (a 5-week exercise-based program of 6 sessions/week (30 sessions)) protocol followed the official ATS/ERS guidelines (Supplementary Information) 86 .In brief, patients undertook a 5-week exercise-based program of 6 sessions/week (30 sessions).Physical exercise was the cornerstone of the program, which also included dietary and psychosocial counselling, based on treadmill walking, stationary cycling, arm ergometry, flexibility, stretching and strengthening exercises with body and fixed weights.The participation was monitored and supervised by a physiotherapist.

EBC collection, NMR sample preparation and spectra acquisition
EBC samples were collected from negativized patients (post-COVID) before entering the rehabilitation program.Control samples were from a cohort of healthy volunteers belonging to an irreversible deidentified set of electronic Maugeri database containing records of people selected from the hospital staff, whose EBC samples

Figure 1 .
Figure 1.Schematic diagram illustrating the overall study design.

Figure 2 .
Figure 2. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) of EBC samples from post-COVID patients and controls.Scores plot showing the degree of separation of the model between post-COVID (red circles) and controls (blue circles).The model presents strong regression (95%, CV-ANOVA p < 2.3 × 10 −12 ) and high-quality parameters (R 2 = 81% and Q 2 = 87%).The labels t[1] and t o [1] along the axes represent the scores (the first 2 partial least-squares components) of the model, which are sufficient to build a satisfactory classification model.

Figure 5 .
Figure 5. Multilevel PLS-DA scores plot for post-COVID patients.The labels X-variate 1 and X-variate 2 along the axes represent the scores (the first 2 partial least-squares components) of the model, which are sufficient to build a satisfactory classification model.Admission variables (IN) are shown in red, while discharge variables (OUT) are in black.

Figure 6 .
Figure 6.Contribution plot of the principal component PC1 of the multilevel PLS-DA model including the clinical parameters of the post-COVID patients.Each bar represents the loading value for each variable on PC1.Admission variables (IN) are shown in red, while discharge variables (OUT) are in black. https://doi.org/10.1038/s41598-023-49601-4

Table 1 .
Characteristics and clinical parameters of the subjects enrolled in the study.IN and OUT refers to post-COVID patients before (IN) and after (OUT) the rehabilitation program.WHO World Health Organization class of severity, BMI body-mass index, TC total cholesterol, TGs triglycerides, AST aspartate aminotransferase, ALT alanine aminotransferase, CRP C-reactive protein, HCO 3 actual bicarbonate, PaO 2 partial pressure of oxygen in arterial blood, PaCO 2 partial pressure of carbon dioxide, FEV 1 forced expiratory volume during the first second of a forced breath, FVC forced vital capacity, FEV 1 /FVC ratio between the forced expiratory volume in the first second (FEV 1 ) and the forced vital capacity (FVC) of the lungs, DLCO carbon monoxide diffusing capacity of the lung, 6MWD six-minute walking distance, CAT chronic obstructive pulmonary disease (COPD) assessment test questionnaire, Barthel index scale used to measure performance in activities of daily living.The values are reported as mean ± SD. a IN/OUT clinical parameters tested with paired Wilcoxon signed ranks test (ns not significant).